Lactic Acid Bacteria

Topics: DNA, Gene expression, RNA Pages: 22 (7813 words) Published: June 22, 2013
Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection Courtney L. Frazier, Joseph San Filippo, Alan M. Lambowitz and David A. Mills Appl. Environ. Microbiol. 2003, 69(2):1121. DOI: 10.1128/AEM.69.2.1121-1128.2003.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2003, p. 1121–1128 0099-2240/03/$08.00 0 DOI: 10.1128/AEM.69.2.1121–1128.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 69, No. 2

Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection Courtney L. Frazier,1 Joseph San Filippo,2 Alan M. Lambowitz,2 and David A. Mills1* Department of Viticulture and Enology, University of California at Davis, Davis, California 95616-8749,1 and Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, Texas 787122 Received 3 October 2002/Accepted 21 November 2002

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Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci. The lactic acid bacteria (LAB) are a broad group of grampositive bacteria that possess similar morphological, metabolic, and physiological characteristics (33). The LAB have been the subject of considerable research and commercial development, given their significance in fermentation, bioprocessing, agriculture, food, and medicine (12, 13). An impediment to fundamental studies on the LAB is the lack of facile genetic tools for manipulation of chromosomal genes (19). Moreover, public concern over the use of genetically engineered cultures for food production has prompted a search for “self-cloning” methods, whereby genetic manipulation is achieved using DNA solely from food-grade microorganisms, preferably from within the same genus (6). Mobile group II introns are catalytic RNA elements present in a wide range of prokaryotic and eukaryotic organisms (18). Some of these introns can mobilize...

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